Part of the work leading to this invention was carried out with United States government support provided under a National Sea Grant Enhancement Grant entitled xe2x80x9cDeveloping a commmercially viable seaweed aquaculture industry in New England.xe2x80x9d Therefore, the U.S. government has certain rights in this invention.
The red alga Porphyra, or nori as it is commonly called, is the most widely eaten and commercially valuable seaweed in the world. Porphyra""s main use is as the purple-black wrapping around the delicacy known as xe2x80x9csushi.xe2x80x9d Nori is commercially grown in Japan, China, Korea and Taiwan, and over 45,000 dry metric tons of nori are produced annually, worth over $2 billion US dollars. Because of its high protein and vitamin content, nori is considered to be a valuable health food. The market for nori sheets in the US alone is estimated to be worth at least $50 million dollars annually and is growing at a rate of over 17% per year. In addition, certain species of Porphyra also serve as important commercial sources of the red pigment r-phycoerythrin, which is utilized as a fluorescent xe2x80x9ctagxe2x80x9d for immunofluorescent studies and can cost as much as $360 per mg.
There are thought to be approximately 70 species of Porphyra worldwide, the majority of which are found in the North Pacific Ocean; approximately 33 species of Porphyra occur in Japan alone. Nori cultivation is a well developed industry, particularly in Japan where it has undergone significant technical improvements since the 1960""s. Improvements made to the technical aspects of nori cultivation include the development of techniques for controlled culturing of its conchocelis stage in shells and for artificial seeding of spores produced by the conchocelis onto cultivation nets which can be stored until placed in the ocean.
As has been demonstrated repeatedly with agricultural crops and other types of cultivation, genetic improvement of cultured species is generally crucial for maximizing yield and developing cost-effective cultivation programs. Seaweeds, including Porphyra, are no exception. However, unlike land plants, seaweed strain improvement techniques have generally been restricted to classical breeding methods, particularly strain selection.
As a result of strain selection efforts, today there are several dozen cultivars of two Porphyra species, P. yezoensis and P. tenera, farmed in Japan. These cultivars were developed primarily as a result of the intensive strain selection program in Japan. Over many years of repeated selection, improvements were made in increasing the average length of fronds, as well as the length of the growing seasons of these two species (see Patwary and van der Meer, 1992). By comparison, efforts to develop new Porphyra strains through sexual hybridization have been far less successful. Intra- and interspecific crosses have been attempted in Porphyra but have contributed little (Suto, 1963). Often the products of sexual crosses have exhibited abnormal growth or chimeric (i.e., sectored) blades. In addition, because most commercially valuable species are monoecious, and thus easily self-fertilized, it is difficult in Poryhrya to produce F1 progeny of specific parents by sexual hybridization.
Thus, the most successful method of producing new strains of Porphyra to date has been through repeated strain selection. However, this approach has a number of disadvantages and limitations. In particular, repeated strain selection usually requires many years of intensive effort and is very labor intensive. In addition, the existing genetic variability in one or more populations of interest may not be sufficient for strain selection purposes. Furthermore, desirable and agronomically-beneficial traits that may be found in other species can not be taken advantage of by applying the methods that have been used in Porphyra in the past. Future improvements in the production of nori, both within and outside of the United States, will therefore most likely depend on the production of new strains that will have to be developed by new strain improvement methods.
One method of strain improvement that permits the rapid development of new strains and the transfer of genes and traits between species is somatic hybridization via protoplast fusion. Protoplast fusion is a well-developed technique in land plants and is just beginning to be successfully applied to seaweeds. In this technique, protoplasts are produced by enzymatically removing the cell walls that surround plant cells, and the protoplasts are then fused together to form a hybrid or a cybrid (i.e., cytoplasmic hybrid). Protoplast fusion can also be used to produce a polyploid or aneuploid. Like sexual hybrids, somatic hybrids generally exhibit combinations of traits found in the two parental plants hybridized. One major advantage of protoplast fusion is that it provides the opportunity to produce unique genomic combinations which are impossible or impractical by sexual hybridization, such as hybridizing individuals of different species and producing cybrids. Protoplast fusion has been reported in only a small number of seaweeds to date, including the green algae Ulva and Enteromorpha (Reddy et al. 1992) and the red algae Gracilaria (Cheney, 1990; Cheney and Duke, 1995), and Porphyra (e.g., Fujita and Migita, 1987; Fujita and Saito, 1990).
Efforts at protoplast fusion in Porphyra date back to 1986, when Saga et al. attempted to fuse protoplasts of a Porphyra species with those of the green alga Enteromorpha without success. Later, Fujita and Migita (1987) reported successfully fusing protoplasts of a wild type strain and a green mutant strain of P. yezoensis using the chemical fusagen polyethylene glycol (PEG). However, although they observed fusion and heterokaryon formation, they ultimately were able to produce only chimeric fronds which in turn produced greenish conchocelis that gave rise to green F1 fronds. In 1990, Fujita and Saito used both PEG and electrofusion techniques in fusion efforts with protoplasts from several Porphyra species. Similarly, Araki and Morishita (1990) attempted to fuse protoplasts between P. yezoensis and P. tenera. Mizukami et al. (1995) used electrofusion to fuse protoplasts between P. yezoensis and P. suborbiculate and report producing xe2x80x9chybrid-likexe2x80x9d thalli. However, none of the fusion studies to date on Porphyra have resulted in a consistent reproducible technique that produces Porphyra strains with stable, improved properties. Effective and practical protoplast fusion methods that can be used for strain improvement in Porphyra would, therefore, be highly desirable.
This invention is directed to a new method for producing wall-less cells, or protoplasts, from the commercially valuable edible seaweed Porphyra (also known as nori) and the use of protoplast fusion techniques for the production of new and improved strains of the same.
The method of the invention features the use of conchoporangial branch conchocelis for at least one of the sources of protoplasts for protoplast fusion. Protoplasts produced from conchosporangial branch conchocelis of one Porphyra species may be mixed with protoplasts produced from blade material, conchocelis or conchospores of a second Porphyra species and fused using, e.g., a chemical fusing agent like polyethylene glycol (PEG) or electrofusion. Other possible fusogens include sodium nitrate, dextran, high pH-high calcium containing solutions or combinations thereof. After fusion has occurred, fusion products are isolated, cultured to multicellular material and regenerated to whole plants. Alternatively, the multicellular material can be used as an undifferentiated cell mass.
Hybrids produced by the method of the invention are expected to possess combinations of the genetic material found in their respective parental species, and, therefore, are expected to exhibit combinations of their traits. Polyploids and aneuploids produced by the method of invention are expected to have one or more extra chromosome in their genome, and, therefore, to have present one or more extra copies of a gene that may code for a desirable trait.
The method of the invention includes a number of improvements over the prior art, which allow for protoplast fusion to be applied to Porphyra in a reproducible and controllable manner and which can result in products that were not possible to produce by prior art methods. For example, the use of conchosporangial branch conchocelis as a source of protoplast for protoplasts fusion simplifies the experiment process as the conchosporangial branch conchocelis phase is easier to grow, subculture and maintain contaminant-free in the laboratory than the blade phase. The term xe2x80x9cconchosporangial branch conchocelis protoplastsxe2x80x9d is defined here to mean protoplasts derived from conchocelis that bear conchosporangial cells. The term xe2x80x9cprotoplastxe2x80x9d refers to a cell in which the cell wall has been enzymatically removed.
We have found that protoplasts produced from conchosporangial branch conchocelis can be fused with protoplasts produced from conchosporangial branch conchocelis of another species, as well as with protoplasts produced from the blades or thallus of another species. Furthermore, we have found that our method produces fusion products that have a higher capacity to regenerate to whole plants than blade-blade protoplast fusion products. In addition, fusion products produced by our method only infrequently result in chimeric blade formation, which is a common occurrence in blade-blade protoplast fusion.
The use of conchosporangial branch conchocelis material as a source of protoplasts for at least one of the parents means that regenerants can be produced that have stable, heritable, new genetic compositions, including hybrid, cybrid, polyploid and aneuploid genomes, useful for strain improvement in the genus Porphyra. Using the method of the invention, we have produced to date several unique plants that show one or more of the following features: 1) unique combinations of plant shape and color; 2) unique combinations of isoenzyme genetic markers; and 3) unique numbers of chromosomes or DNA contents. These plants may be either hybrids, cybrids, polyploids or aneuploids; any of these examples could represent an improved strain. Because both polyploids and hybrids in particular have been shown to be valuable cultivars for many land crop species, this discovery provides support for our methods for protoplast fusion being a useful and practical approach for strain improvement in Porphyra.
Therefore, the invention also features new varieties of Porphyra produced according to the method of the invention. In particular, the invention is directed to a variety of Porphyra, having the nuclear 18s rDNA sequence of P. yezoensis as found in GenBank Accession No. D79976, and furthermore having 4 or more chromosomes, preferably 4-6 chromosomes, (haploid number) per cell; or to a variety of Porphyra, having the nuclear 18s rDNA sequence of P. umbilicalis as found in GenBank Accession No. L36049, and furthermore having 5 or more chromosomes, preferably 5-8 chromosomes, (haploid number) per cell.
In addition, the invention is directed to a variety of a Porphyra species, selected from the group consisting of a variety having the nuclear 18s rDNA sequence of P. haitanensis and having 6 or more chromosomes (haploid number) per cell, a variety having the nuclear 18s rDNA sequence of P. kuniedae and having 3 or more chromosomes (haploid number) per cell, a variety having the nuclear 18s rDNA sequence of P. leucosticta and having 5 or more chromosomes (haploid number) per cell, a variety having the nuclear 18s rDNA sequence of P. linearis and having 5 or more chromosomes (haploid number) per cell, a variety having the nuclear 18s rDNA sequence of P. pseudolinearis and having 5 or more chromosomes (haploid number) per cell, a variety having the nuclear 18srDNA sequence of P. purpurea and having 6 or more chromosomes (haploid number) per cell, a variety having the nuclear 18srDNA sequence of P. suborbiculata and having 3 or more chromosomes (haploid number) per cell, and a variety having the nuclear 18srDNA sequence of P. tenera and having 4 or more chromosomes (haploid number) per cell.